Compositions and methods for RNA-based antimicrobial drug targeting

ABSTRACT

A ribonucleic acid (RNA) based modular regulatory element (MRE) for inhibiting translation of a target gene transcript in an organism (e.g. a pathogen) includes a first segment of RNA capable of forming a stem loop structure, a second segment of RNA downstream of the first segment of RNA and having a target sequence capable of binding to the target gene transcript (or polycistron lead gene transcript), and a third segment of RNA downstream of the second segment of RNA and being capable of forming a terminator hairpin downstream of the target sequence. Methods for assaying the role of a putative target gene in an organism include inhibiting expression of various target genes using the RNA-based MRE.

CROSS-REFERENCE TO RELATED APPLICATION(S)

The present application claims priority to and the benefit of U.S.Provisional Application Ser. No. 62/452,217 filed on Jan. 30, 2017,entitled “COMPOSITIONS AND METHODS FOR RNA-BASED ANTIMICROBIAL DRUGTARGETING,” the entire content of which is incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The United States government has certain rights in this inventionpursuant to Contract No. DE-AC52-06NA25396 between the United StatesDepartment of Energy and Los Alamos National Security, LLC for theoperation of Los Alamos National Laboratory.

INCORPORATION BY REFERENCE

The present application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 29, 2018, isnamed 147368SEQLISTING.txt and is 5,083 bytes in size.

BACKGROUND

The majority of currently used antibiotics are compromised by theemergence of multiple drug resistance (MDR) mechanisms. Antimicrobialresistance is an unavoidable effect of continuous use of antibioticsrendering humans and animals vulnerable to MDR. It has been postulatedthat some parts of the world are in, or could soon be in, apost-antibiotic era. At this time, there are fewer than 40 proteintargets for antimicrobial treatments, and unfortunately, there is a lackof effective methods to identify and characterize novel or uniquetherapeutic targets.

SUMMARY

In some embodiments of the present invention, a modular regulatoryelement (MRE) composition is composed of a ribonucleic acid (RNA) thatinhibits translation of a target gene transcript. The MRE composition aspresented in the present disclosure may also be referred to as an “RNARegulator,” a “Riboregulator”, or “Riboregulator RNA.” This RNA includesa first segment of RNA capable of forming a stem loop structure, asecond segment of RNA downstream of the first segment of RNA andincluding a targeting sequence capable of binding to the target genetranscript, and a third segment of RNA downstream of the second segmentof RNA and being capable of forming a terminator hairpin downstream ofthe targeting sequence. In some embodiments, the targeting sequence iscapable of binding to a ribosomal binding site (RBS) of the target genetranscript. In some embodiments, the targeting sequence is complementaryto the RBS of the target gene transcript.

In some embodiments of the present invention, a method of assaying theessentialness of a putative target gene in an organism (e.g., apathogen) includes culturing the organism expressing the modularregulatory element (MRE), and binding the MRE to the ribosomal bindingsite (RBS) of a transcript of the putative essential gene to inhibittranslation of the putative essential gene. As used herein, an“essential gene” refers to a gene that is required for growth of theorganism and includes absolute essential genes and conditional essentialgenes. In some instances, the essential gene is an absolute essentialgene as the gene is required for growth of the organism in anycondition. In other instances, the essential gene is a conditionalessential gene and is required for growth of the organism in thepresence of an additional factor such as an antibiotic. In someembodiments, binding the MRE to the RBS of a transcript of the putativeessential gene includes expressing the modular regulatory element (MRE)composition for inhibiting translation of a target gene transcript asdescribed herein. In some embodiments, the putative essential gene inthe organism is not found in humans (e.g., no homologs of the pathogengene are found in humans). In some embodiments, the method of assaying aputative essential gene in an organism also includes adding anantibiotic to a culture of the organism either prior to or after thebinding of the MRE to the RBS of the transcript. In some embodiments ofthe present invention, the activity of the MRE is determined bymonitoring the growth of the organism or by high throughput sequencing.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a flow chart diagram depicting the modular regulatory element(MRE) design and production, including the identification of a targetsequence for a selected organism (organism A) using comparativealignment with organism B, according to embodiments of the presentinvention.

FIG. 2A is a schematic of an RNA-based modular regulatory element (MRE)(Riboregulator RNA (5)) having a 5′ RNA stem loop (10), a variabletarget sequence (15) for binding to the ribosomal binding site (RBS)(30) on a putative target gene transcript (25) in an organism, and anRNA terminator hairpin sequence (20), according to embodiments of thepresent invention.

FIG. 2B is a schematic of the RNA Regulator (5) of FIG. 2A in its boundstate to the RBS of the putative target gene transcript (25), therebyoccluding translation of the putative target gene, according toembodiments of the present invention.

FIG. 3 is a flow diagram of the methods for targeting putative essentialgenes in organisms (e.g., Burkholderia thailandensis (B. thailandensisor Bt)) by assessing normal versus compromised growth of the organismwhen translation of a putative target gene is inhibited using an MRE (5)followed by addition of a selected antibiotic to Bt culture showingnormal growth upon inhibition of a putative target gene in order toscreen for antimicrobial resistance genes, according to embodiments ofthe present invention.

FIG. 4A is a schematic of the pMo168 plasmid vector having amobilization element for transfer into a target organism by tri-parentalmating having the specific MRE sequence for targeting the AmrA gene(Amr_(AMRE)) driven by the strong P1 promoter for constitutiveexpression in B. thailandensis (Bt), and a schematic of the pSCPrhaB2plasmid having a modular regulatory element (MRE) for targeting the AmrAgene (Amr_(AMRE)) under the control of the rhamnose inducible promoter(PrhaB), according to embodiments of the present invention.

FIG. 4B shows a left graph depicting the growth of non-pathogenicBt-E264 in the presence of gentamicin at the indicated minimuminhibitory concentration (MIC) μg/mL, without an RNA Regulator (pMOempty vector, green) and with the pMO168-Amr_(AMRE) plasmid(pMo+Amr_(AMRE), no growth (i.e., no maroon)) as shown in FIG. 4Aconstitutively expressing a target sequence that inhibits the AmrA genetranslation, and a right graph depicting the growth of Bt-E264transformed with the pSCPrhaB2 Amr_(AMRE) plasmid as shown in FIG. 4A inthe presence of gentamicin at the indicated minimum inhibitoryconcentration (MIC) μg/mL, in the presence of 0.25% rhamnose (green), norhamnose (maroon), or 0.5% glucose (orange), according to embodiments ofthe present invention.

FIG. 4C is a graph of the growth of the pathogenic Bt-CDC272 in thepresence of gentamicin at the indicated minimum inhibitory concentration(MIC) ug/mL, without an RNA Regulator (pMO empty vector, green) and withthe pMO168-Amr_(AMRE) plasmid (pMo+Amr_(AMRE), low growth observed inmaroon on right) constitutively expressing a target sequence thatinhibits the AmrA gene translation, according to embodiments of thepresent invention.

FIG. 5A is a schematic of the pSCPrhaB2 plasmid vector having amobilization element and a rhamnose inducible promoter (PrhaB) forinducing expression of the specific MRE sequence (iRec_(AMRE)) fortargeting the RecA gene in the presence of rhamnose and constitutiveexpression of the cAmr_(AMRE) RNA Regulator for constitutive inhibitionof the AmrA gene translation, according to embodiments of the presentinvention.

FIG. 5B is a graph of the growth of Bt-E264 in the presence ofciprofloxacin at the indicated minimum inhibitory concentration (MIC)μg/mL, without a plasmid (empty vector, green shown on left), with thepSCPrhaB2-cAmr_(AMRE)-iRec_(AMRE) plasmid of FIG. 5A in the presence ofglucose only (−rham+gluc, shown in maroon), and in the presence ofglucose and rhamnose (+0.25% rham+0.5% glu, minimal growth shown inorange), according to embodiments of the present invention.

FIG. 6 is a schematic of the pSCPrhaB2 plasmid having a modularregulatory element (MRE) cassette (orange) for insertion andtranscription of a selected RNA Regulator sequence under the control ofthe rhamnose inducible promoter (PrhaB)(green), according to embodimentsof the present invention.

FIG. 7A is a schematic depicting method steps for a novel drug targetingscreening of a library of MREs in B. thailandensis, including mating,growth, and sample collection process for screening and subsequent deepsequencing, according to embodiments of the present invention.

FIG. 7B is a graph plotting the fraction of the population relative tothe number of MREs (Riboregulators) at day 0 (green), day 1 (blue), andday 13 (red) of the library screen of FIG. 7A, according to embodimentsof the present invention.

FIG. 8A shows data plots tracking the relative abundance growth in Btfor 500 different MREs as a function of time for a Bt MRE library inwhich the upper plot shows the relative abundance growth of Bt in thepresence of ampicillin and no rhamnose and the lower plot shows therelative abundance growth of Bt in the presence of ampicillin andrhamnose, according to embodiments of the present invention.

FIG. 8B shows data plots tracking the relative abundance growth in Btfor 500 different MREs as a function of time for a Bt MRE library inwhich the upper plot shows the relative abundance growth of Bt in thepresence of gentamicin and no rhamnose, and the lower plot shows therelative abundance growth of Bt in the presence of gentamicin andrhamnose, according to embodiments of the present invention.

FIG. 8C is a graph plotting the data from FIGS. 8A and 8B as targets ofputative essential genes or polycistronic lead genes, in which the barplots represent the effects of the MREs of the individual putativeessential genes (values less than 0) or when targeted as part of apolycistron (values greater than 0), according to embodiments of thepresent invention.

DETAILED DESCRIPTION

Multiple drug resistant (MDR) pathogens may be defeated by at least oneof two main paths: 1) identification of novel antimicrobial drugs andtherapies; and/or 2) deactivation of the current MDR mechanisms, therebymaking the pathogen susceptible to existing antibiotic therapies. Someembodiments of the present invention are directed to the inhibition ofgenes that are essential for the MDR pathogen or the MDR mechanism inthe pathogen.

For example, embodiments of the present invention include methods foridentifying putative target genes in a selected organism (e.g., apathogen) testing the putative target gene by deactivating theexpression of the gene. More specifically, embodiments of the presentinvention include methods for deactivating a putative essential targetgene by binding the ribosomal binding site (RBS) of the putativeessential target gene transcript, and thereby blocking translation.

Also, as used herein, the term “transcript” refers to the messengerribonucleic acid (mRNA) formed from transcription of the gene. As usedherein, “deactivation,” “deactivating,” and like terms in reference to agene refer to inhibition of the mRNA translation of the gene's mRNA toprotein. Deactivation of a gene by inhibition of a gene's mRNAtranslation is also referred to as “inhibition of expression,”“inhibition of gene expression,” “inhibition of translation” and “MREactivity”. As used herein, “heterologous expression” refers to theexpression nucleic acid vectors (e.g., DNA plasmid vectors) that areintroduced and are exogenous to the host organism.

As used herein, “putative target gene,” putative essential gene,”“putative essential target gene,” and “target gene,” are usedinterchangeably to refer to the particular candidate gene that is thebinding target of the MRE to be assayed for its role in the growth ofthe organism either under normal growth conditions or in the presence ofan antibiotic.

In some embodiments, methods of screening for multidrug resistance (MDR)mechanisms include testing a putative target gene by inhibitingexpression of the target gene in the presence of an antibiotic todetermine the effect of the inhibited gene on MDR resistance and/orbacterial physiology in the presence of the antibiotic.

With reference to FIG. 1, in some embodiments of the present invention,a methodology for identifying and validating putative target genes in atarget pathogen (organism B) includes determining a target sequence ofthe organism (Organism B) in silico by aligning all of the essentialgenes or genes known to be essential for multidrug resistance of a knownselected organism designated as Organism A. Organism A is acharacterized organism for which essential gene research has beenpreviously performed (or suggested) and a list of essential genes areidentified in an essential gene database. Accordingly, in someembodiments, the known essential genes of Organism A are aligned with adetermined sequence of Organism B (the target organism) to retrieve theRBS sequences from all of the essential genes that are homologous (i.e.,those with at least 70% identity) between Organisms A and B.Accordingly, the RBS sequences of the targeted essential genes are knownas the “target sequences” and are more distinctly referred to as the RBSof the gene transcript, whereas the “targeting sequence” is thehybridizing RNA sequence that binds to the target sequence (e.g., theRBS) on the targeted gene transcript. In some embodiments of the presentinvention, if the putative essential gene is in a polycistron, then thelead gene in the polycistron is identified along with the correspondingRBS sequence for the lead gene. In some embodiments, the targetingsequence includes an RNA sequence that hybridizes to the RBS sequence ofthe targeted gene or another gene within the polycistron. In someembodiments, the targeting sequence also includes a flanking sequencethat contains specific restriction sites used for direct cloning of thetargeting sequence into the MRE. Accordingly, the targeting sequencesfor binding the putative essential genes of a target pathogen (OrganismB) are identified based on comparative alignment of the essential geneswith organism A.

With reference to FIGS. 2A and 2B, according to embodiments of thepresent invention, a ribonucleic acid (RNA) composition for inhibitingtranslation of a putative (candidate) target gene is the RNA-basedmodular regulatory element (MRE) having a targeting sequence for bindingto the ribosomal binding site (RBS) of a putative essential gene or agene required for antimicrobial resistance (AMR) in an organism (e.g., apathogen). This binding of the targeting sequence to the RBS of thetarget gene transcript inhibits expression of the target genetranscript.

According to embodiments of the present invention, a ribonucleic acid(RNA) composition for inhibiting translation of a target genetranscript, includes a first segment of RNA capable of forming a 5′ stemloop structure (10), a second segment of RNA downstream of the firstsegment of RNA and having a targeting sequence (15) capable of bindingto the target gene transcript (25), and a third segment of RNAdownstream of the second segment of RNA that forms a 3′ terminator (20)downstream of the targeting sequence.

With reference to FIG. 2A, an RNA Regulator (5) is an RNA compositionthat is a type of RNA-based MRE according to embodiments of the presentdisclosure. In some embodiments of the present invention, the RNARegulator (which may also be referred to herein as a Riboregulator) isan RNA-based MRE made of a contiguous (e.g., consecutive) RNA sequencehaving a least three RNA segments. The first RNA segment is a 5′ RNAstem loop (10), the second RNA segment is a targeting sequence (15), andthe third RNA segment is a 3′ terminator hairpin (20). While eachsegment of RNA may be characterized from another, the segments of RNAare part of one transcribed sequence unit. In some embodiments, thethree segments of RNA may be separated by a flanking RNA sequence toprovide spacing, and/or to allow for replacement of the segments of theRNA Regulator and/or to allow for insertion of the segments into anexpression plasmid. The flanking RNA sequence may be referred to as aspacer RNA. In addition to flanking RNA sequences (or spacer RNA), theRNA segments may be separated by restriction site sequences therebyallowing for a segment to be enzymatically cut out of the contiguoussequence.

According to some embodiments of the present invention, an RNA Regulator(5) includes a strong 5′ RNA stem-loop (e.g., ΔG°<=−40 kcal/mol) and anefficient terminator (20) which together bracket the variable targetingsequence (15). As used herein, a 5′ RNA stem loop includes any RNAsequence that forms a stem loop and has a free energy that is not morethan about 54 kcal/mol. In some embodiments of the present invention,the 5′ RNA stem loop (10) of the RNA Regulator (5) is SEQ ID NO: 1AUUCGAGCCUCUCCUUCUAUCGGCGUGUGACGAGAAAUCGUAAUGCGUCGAUA GAAGGAGAGGUUCGAAUor SEQ ID NO: 2 UAAGCUUGGAGAGGAAGAUAGCUGCGUAAUGCUAAAGAGCAGUGUGCGGCUAUCUUCCUCUCCGAGCUUA.

In other embodiments, the RNA Regulator (5) includes a 5′ RNA stem loop(10) having a smaller helix structure than the 5′ RNA stem loop (10) ofSEQ ID NO: 1 or SEQ ID NO: 2 so long as the smaller helix has a freeenergy that is not more than about 54 kcal/mol. For example, the 5′ RNAstem loop (10) may form a helix structure that is a helix of 23 pairedRNA basepairs (“helix-23”) or a helix of 12 paired RNA basepairs(“helix-12”). In some embodiments, the 5′ RNA stem loop (10) is ahelix-23 of SEQ ID NO: 9AUUCGAGCCAUCGGCGUGUGACGAGAAAUCGUAAUGCGUCGAUGGUUCGAAU or SEQ ID NO: 10UAAGCUCGGUAGCCGCACACUGCUCUUUAGCAUUACGCAGCUACCAAGCUUA. In otherembodiments, the 5′ RNA stem loop (10) is a helix-12 of SEQ ID NO: 11AUUCGAGCCAUAGAAAUAUGGUUCGAAU or SEQ ID NO: 12UAAGCUCGGUAUCUUUAUACCAAGCUUA.

As used herein, the “targeting sequence” (15) of the RNA Regulatorincludes an RNA sequence that hybridizes to a region in the mRNAcentered on the ribosome binding site (RBS) of the putative target gene.That is, the targeting sequence (15) for a putative essential gene in anorganism includes the complementary RNA sequence that will hybridize toan mRNA sequence that spans from a position at least 20 nucleotide bases5′ (upstream) of the RBS translational start site to a position at least20 bases 3′ (downstream) to the RBS translational start site. Thetargeting sequences are complementary to and overlap the region spanningthe ribosomal binding site (RBS) in a targeted essential gene's mRNA.The specific interaction of the RNA Regulator with the mRNA preventstranslational initiation (e.g., ribosome binding), effectivelyabrogating expression.

As discussed herein, the RNA Regulator (5) also includes a 3′ terminatorhairpin (20), also referred to herein as a 3′ terminator (20). As usedherein, the 3′ terminator or 3′ terminator hairpin (20) includes anysuitable RNA terminator hairpin for the target organism. In someembodiments, the 3′ terminator (20) has an RNA sequence of SEQ ID NO: 3AUCAAUAAAACGAAAGGCUCAGUCGAAAGACUGGGCCUUUCGUUUUAUCUGUUG

With reference to FIG. 2B, binding of the targeting sequence (15) of theRNA Regulator to the cognate RBS (30) on the targeted mRNA genetranscript (25) occludes the RBS from being bound by the ribosome forinitiation of translation thereby inhibiting gene expression.

The components of the RNA Regulator (e.g., the 5′ stem loop, the targetsequencing and the 3′ terminator) are modular, allowing for easyexchange of the targeting sequences using unique restriction sites inthe MRE and Gibson assembly techniques. In some embodiments of thepresent invention, the components of the RNA Regulator are designed as acassette for insertion into the pMO168 vector containing a mobilizationelement for transfer into a target organism by tri-parental mating, thuscreating pMO168-MRE as depicted in FIG. 4A. In some embodiments of thepresent invention, a DNA vector encodes for an RNA Regulator having a 5′stem loop (5) of SEQ ID NO: 6 and a 3′ terminator (20) of SEQ ID NO: 10.

In some embodiments, a DNA vector encodes for an RNA Regulator having a5′ stem loop (5) with helix-23 encoded by SEQ ID NO: 13ATTCGAGCCATCGGCGTGTGACGAGAAATCGTAATGCGTCGATGGTTCGAAT or SEQ ID NO: 14TAAGCTCGGTAGCCGCACACTGCTCTTTAGCATTACGCAGCTACCAAGCTTA, together with a 3′terminator (20) of SEQ ID NO: 7.

In some embodiments, a DNA vector encodes for an RNA Regulator having a5′ stem loop (5) with helix-12 encoded by SEQ ID NO: 15ATTCGAGCCATAGAAATATGGTTCGAAT or SEQ ID NO: 16TAAGCTCGGTATCTTTATACCAAGCTTA, together with a 3′ terminator (20) of SEQID NO: 7.

In order to engineer an MRE to identify and validate putative essentialgenes that are critical to an organism's survival and multi drugresistance (MDR), the model pathogen Burkholderia pseudomallei wasselected as organism A. The B. pseudomallei pathogen has 312 proteinencoding genes that are not found in humans and have beencomputationally predicted to be essential for its survival andpathogenicity, as described in Chong et al., 2006, “In silico analysisof Burkholderia pseudomallei genome sequence for potential drugtargets,” In Silico Biol 6:341-346, the entire content of which isincorporated herein by reference. For validation of the 312 putativeessential genes of B. pseudomallei, the non-pathogenic surrogate,Burkholderia thailandensis was used as the target organism, organism B.

In order to assay the MRE for multi-drug resistance, an MDR assay wasfirst validated in the B. thailandensis (Bt) target organism. The AmrABmultidrug efflux pump in Bt is known to confer resistance to theantibiotic gentamicin. By deactivating expression of the Bt-AmrA gene,the AmrAB efflux pump is not formed, rendering Bt susceptible togentamicin. Accordingly, a vector (pMo168) as depicted in FIG. 4A wascreated allowing for the constitutive expression of an RNA Regulatortargeting the RBS of the AmrA gene transcript (Amr_(AMRE)). ThispMo168-Amr_(AMRE) plasmid was transformed into Bt-E264 cells, and theresulting strain showed inhibited growth in the presence of gentamicinas shown in FIG. 4B. Accordingly, the Amr_(AMRE) MRE is effective forinhibiting gene expression of the AmrA gene and increasingsusceptibility of Bt-E264 cells to gentamicin.

The Amr_(AMRE) was also expressed in a pSCPrhaB2 plasmid as depicted inFIG. 4A under a rhamnose-inducible promoter. This pSCPrhaB2-AmrAMREplasmid was also transformed into Bt-E264 cells in the presence andabsence of rhamnose. In the pSCPrhaB2-Amr_(AMRE) plasmid, the PrhaBrhamnose inducible promoter controls the expression of the Amr_(AMRE)MRE, and the results in FIG. 4A show that the PrhaB promoter is not ableto fully control (e.g., shutdown) the expression of the Amr_(AMRE) MREin the absence of rhamnose and that the addition of 0.50% glucose helpsthe promoter to better control (e.g., shutdown) the expression of theAmr_(AMRE) MRE in the absence of rhamnose. Additionally, as observed inFIG. 4B, the level of expression of the Amr_(AMRE) MRE caused by theleakiness of the PrhaB promoter is sufficient to restore susceptibilityof Bt-E264 to gentamicin.

The RecA and AmrA genes were previously identified as co-resistancemechanisms against ciprofloxacin treatment. RecA is responsible foractivating the repair of the DNA that is cleaved by the action ofciprofloxacin, and AmrA is part of the multidrug efflux pump, asdescribed respectively in Thi et al., 2011, J. Antimicrob Chemother,“Effect of recA inactivation on mutagenesis of Escherichia coli exposedto sublethal concentrations of antimicrobials,” 66: 531-538; andPodnecky et al., 2015, Front Microbiol., “Efflux pump-mediated drugresistance in Burkholderia,” 6:305, the entire contents of both of whichare incorporated herein by reference. For the MDR resistance assay, avector as depicted in FIG. 5A was created allowing for the simultaneousconstitutive expression of an MRE targeting the RBS of the AmrA genetranscript (Amr_(AMRE)) and an MRE targeting the RBS of RecA(Rec_(AMRE)) under a rhamnose inducible promoter.

For validation of the predicted putative essential genes, a library of500 MREs was constructed having a targeting sequence for each of theidentified putative essential genes. Some of the 312 putative essentialgenes (described in Chong et al., 2006, supra) were found within apolycistron, and in such cases an MRE was made with a targeting sequence(15) to the putative gene and a second MRE was made with a targetingsequence (15) to the lead gene of the polycistron. The addition of theMREs targeting the polycistron lead genes increased the MRE library from312 to 500. The library of vectors containing the 500 MREs wasconstructed and transformed into Bt for culturing.

In some embodiments of the present invention, a method of assaying aputative essential gene in an organism includes culturing the organismand binding the ribosomal binding site (RBS) of a transcript of theputative essential gene to thereby inhibit translation of the putativeessential gene. In some embodiments, a method of assaying a putativeessential gene in an organism includes culturing the organism, andbinding the ribosomal binding site (RBS) of a transcript of the leadgene of a polycistron gene cluster that includes the putative essentialgene to thereby inhibit translation of the putative essential gene. Insome embodiments, the binding of the RBS of a transcript of the putativeessential gene includes transforming the organism with a vectorexpressing an RNA composition (RNA Regulator)(5) targeting the RBS ofthe putative essential gene or targeting the RBS of the lead gene of thepolycistron gene cluster. The putative essential gene is a gene notfound in humans. In some embodiments, the method of assaying includesinducing expression of the RNA Regulator (5). In some embodiments, themethod of assaying includes inducing expression of the RNA Regulator (5)in the presence of an antibiotic. In some embodiments, the antibiotic isadded to the organism culture either prior to or after the binding ofthe ribosomal binding site (RBS) of the transcript of the putativeessential gene or lead gene of the polycistron gene cluster.

With reference to FIG. 6, each of the specifically constructed RNARegulators (5) was separately cloned into a pSCPrhaB2 plasmid withtranscription induced in the presence of rhamnose. The transformationand screening (i.e. selection) of this library of 500 RNA Regulators inBt is shown in FIG. 7A with the relative abundance growth plots underthe selective conditions shown in FIGS. 8A and 8B. The bar plot in FIG.8C plots the data from FIGS. 8A and 8B showing that for some targetgenes, targeting the lead gene of a polycistron is required toknock-down or inhibit expression.

The following Examples are presented for illustrative purposes only, anddo not limit the scope or content of the present application.

EXAMPLES Example 1

Materials and Methods. Vector PMo168 is described in Hamad et al., Gene,2009, 430:123-31, the entire content of which is incorporated herein byreference, and vector pSCPrhaB2 is described in Cardona et al., Plasmid,2005, 54:219-28, the entire content of which is incorporated herein byreference.

Example 2

RNA Regulator (5) Sequences. For transcription of the RNA Regulators,DNA constructs were synthesized for plasmid expression. The complete DNAsequence for the AmrA RNA Regulator construct for insertion into vectorpSCPrhaB2 or pMo168 is (SEQ ID NO: 4)GAATTCCATTCGAGCCTCTCCTTCTATCGGCGTGTGACGAGAAATCGTAATGCGTCGATAGAAGGAGAGGTTCGAATTATACATGTTATCAGCGCATGCGTGCCCATTCGTATTTCATCGTTTTCCTCGCAAGTCGCTCGACCGGGACGAATTGCGATCGCGATATCAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGCTGCAG CTCTAGA.

The SEQ ID NO: 4 AmrA DNA construct is made up of: an EcoRI restrictionsite GAATTC a spacer of one nucleotide (C), a 5′-Stem loop (10) (SEQ IDNO: 5), ATTCGAGCCTCTCCTTCTATCGGCGTGTGACGAGAAATCGTAATGCGTCGATAGAAGGAGAGGTTCGAAT a spacer of 3 nucleotides (TAT), an PciI restriction siteACATGT an AmrA targeting sequence (15) (SEQ ID NO: 6),TATCAGCGCATGCGTGCCCATTCGTATTTCATCGTTTTCCTCGCAAGTCGCTCGAC CGGGACGAATT anAsisI restriction site GCGATCGC a spacer of 3 nucleotides (GAT),a 3′Terminator (SEQ ID NO: 7),ATCAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTG a spacer of 7nucleotides (CTGCAGC) and a XbaI restriction site TCTAGA

Example 3

Design of a Modular Regulatory Element (MRE) Targeting GentamicinResistance (AmrA_(MRE) or AmrA_(MRE)). AmrA is a periplasmic proteinthat links the transmembrane domains of the AmrAB-OprA pump that isresponsible for gentamicin antibiotic resistance as well as resistanceto other antibiotics. The initial cassette contains an MRE targetingsequence for AmrA (Amr_(AMRE))(SEQ ID NO: 4) driven by the strong P1promoter for constitutive transcription in Burkholderia thailandensis.

AmrA_(MRE) P1 promoter cassette SEQ ID NO: 8:GATATCGAGACGAACCCAGTTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTAGCCTCGGGCATCCAAGCTAGCTAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAACAGCAACGATGTTACGCAGCAGGGTAGTCGCCCTAAAACAAAGTTAGGCAGCCGTTGTGCTGGTGCTTTCTAGTAGTTGTTGTGGGGTAGGCAGTCAGAGTTCGATTTGCTTGTCGCCATAATAGATTCACAAGAAGGATTCGACATGGGTCAAAGTACATTCGAGCCTCTCCTTCTATCGGCGTGTGACGAGAAATCGTAATGCGTCGATAGAAGGAGAGGTTCGAATTATACATGTTATCAGCGCATGCGTGCCCATTCGTATTTCATCGTTTTCCTCGCAAGTCGCTCGACCGGGACGAATTGCGATCGCGATATCAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGCTGCAGCGATATC.

Example 4

Design of Modular Regulatory Elements targeting ciprofloxacin resistance(RecA_(MRE)/RecA_(MRE) and AmrA_(MRE)/AmrA_(MRE)). The RecA and AmrAgene products have been identified as co-resistance mechanisms againstciprofloxacin treatment. RecA is responsible for activating the repairof the DNA that is cleaved by the action of ciprofloxacin and AmrA ispart of the multidrug efflux pump. A vector was created allowing for,simultaneously, the constitutive expression of the AmrAMRE and aninducible expressed MRE targeting RecA (RecA_(MRE)). Here the targetingsequence for AmrA was swapped for one targeting RecA in thepSCPrhaB2-MRE. The constitutive P1-AmrA_(MRE) cassette was then insertedat a second site in the vector creating pSCPrhaB2-MRE₂ as shown in FIG.5A with a RecAMRE sequence of (SEQ ID NO: 17):GAATTC-C-ATTCGAGCCTCTCCTTCTATCGGCGTGTGACGAGAAATCGTAATGCGTCGATAGAAGGAGAGGTTCGAATTATACATGTTATCACAGCCCGGAGCCTTTCTTGCTTTCTTCCATGAATCGTCCTTTGCTATGATGAGCAGCGTCTTGCGATCGCGATATCAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGCTGCAGCTCTAGA

Example 5

Design of Modular Regulatory Elements targeting putative essential genesin Burkholderia thailandensis (Bt). A list of the putative essentialgenes from Burkholderia pseudiomallei (Bp) was obtained from Chong etal. 2006, supra. The list of genes from Bp was converted to a list ofhomologs in Bt using a custom software program, but could be obtainedusing the Basic Local Alignment Search Tool (BLAST). Targeting sequencesthat bind to the ribosomal binding site (RBS) of each putative gene wereextracted automatically from the genomic sequence. These extractedtargeting sequences are then synthesized and combined to create alibrary of RNA Regulators (MREs) specific for each of the putativeessential genes. This MRE library was then inserted into the pSCPrhaB2plasmid shown in FIG. 6 using Gibson assembly.

Example 6

Incorporation of MRE expression vectors into Bt. A modification of thetriparental mating method developed for Burkholderia gladioli asdescribed in Somprasong et al., 2010 was used to transform theexpression vectors into Bt. (Somprasong et al., 2010, “Methods forgenetic manipulation of Burkholderia gladioli pathovar cocovenenans,”BMC Res Notes, 3:308, the entire content of which is incorporated hereinby reference.) Briefly, 8 mls of the following strains were grownovernight: Bt E264 (LB broth, carbenicillin 50 μg/ml; E. coli DH5-αcells containing the triparental Helper vector (LB, kanamycin 50 μg/ml);and E. coli DH5-α cells containing pSCPrhaB2-MRE (LB, trimethoprim 50μg/ml. The outgrowth was approximately equivalent for all cultures andeach culture of cells was pelleted and washed 3 times with LB broth andresuspended in a final volume of 200 μl. 80 μl of the donor and helperwere then combined and 80 μls of the mix was transferred to the Bt tubeand mixed. The samples were then placed at 30° C. for 1 hour and thecontents were spread on LB plates without antibiotic and incubatedovernight at 30° C. Plates were then scraped with 2 mls of LB. 25 μlfrom each scraped culture were plated (LB trimethoprim 50 μg/ml,gentamicin 50 μg/ml to assess the mating efficiency. These test platesshowed no growth for the Donor/helper control. The remainder of the 2 mlmating sample was then transferred to 100 ml LB with trimethoprim 50μg/ml and gentamicin 50 μg/ml and grown overnight at 30° C.

Example 7

Activity Screening of MREs. MREs targeting the gentamicin andciprofloxacin antibiotic resistance mechanisms were screened foractivity by monitoring their effect on Bt growth in the presence of theantibiotics.

Activity screening for AmrA_(MRE). The screening of the activity of theconstitutively expressed and inducible versions of the AmrA_(MRE)against gentamicin resistance was performed by incubating AmrA_(MRE)expressing B. thailandensis overnight in the presence of increasingconcentrations of gentamicin, from 0 to 400 μg/mL as shown in FIG. 4B.In addition the constitutive AmrA_(MRE) was also tested with thepathogenic version of B. thailandensis (Bt-CDC272) and the results asshown in FIG. 4C were comparable to those of the non-pathogenic Bt.

Activity screening for RecA_(MRE)/AmrA_(MRE). The screening of theactivity of the constitutively expressed AmrA_(MRE) and inducibleRecA_(MRE) against ciprofloxacin resistance was performed by incubatingRecA_(MRE)/AmrA_(MRE) expressing B. thailandensis overnight in thepresence of increasing concentrations of ciprofloxacin, from 0 to 100μg/mL. The expression of the RecAMRE was induced by the addition of 0.2%rhamnose as shown in FIG. 5B.

Activity screening for the Bt MRE library targeting all putativeessential genes. The screening of the activity of the inducibleexpressed Bt MRE library was performed by incubating the Bt MRE libraryexpressing B. thailandensis for 13 days in the absence and presence of0.2% rhamnose as shown in FIG. 7A. AmrA was used as the positive controlfor this experiments and the Bt MRE library (containing AmrA_(MRE))expressing B. thailandensis was also incubated with and withoutgentamicin to confirm the activity of the MREs, specifically theactivity of the AmrA_(MRE) as shown in FIGS. 8A and 8B and then plottedas targeting a putative essential gene or polycistronic lead gene asshown in FIG. 8C. After transformation of the MRE library into Bt cells,the cells were grown overnight and four large 500 mL cultures wereinoculated with the cells and supplemented with the rhamnose andgentamicin as described in the above conditions. Aliquots (8 mL) werecollected at the specific intervals (Day 1, 5, 6, 8 and 13) and thevectors containing the MRE targeting genes in the library were purifiedby Mini-Prep and PCR barcoded by day and condition. The PCR productsfrom each day and condition were collected, quantified and mixed,followed by deep sequencing using an IIlumina miSeq platform. Data wereanalyzed using a custom software program to extract, identify, andquantify the targeting sequences from each time point and condition.FIG. 7A illustrates the mating (transformation, steps 1 to 5) and samplecollection process and FIG. 7B shows an increase in the fraction of thepopulation with MRE targeting genes over the 13 day time course

While the present invention has been illustrated and described withreference to certain exemplary embodiments, those of ordinary skill inthe art will understand that various modifications and changes may bemade to the described embodiments without departing from the spirit andscope of the present invention, as defined in the following claims.

What is claimed is:
 1. A ribonucleic acid (RNA) composition forinhibiting translation of a bacterial target gene transcript, the RNAcomposition comprising: a first segment of RNA capable of forming a 5′stem loop structure, the first segment of RNA comprising a sequence ofSEQ ID NO: 1, 2, 9, 10, 11, or 12; a second segment of RNA downstream ofthe first segment of RNA, the second segment comprising a targetingsequence capable of binding to a ribosomal binding site (RBS) of thebacterial target gene transcript; and a third segment of RNA downstreamof the second segment of RNA, the third segment capable of forming a 3′terminator downstream of the targeting sequence, the third segment ofRNA comprising a sequence of SEQ ID NO:
 3. 2. The RNA composition ofclaim 1, wherein the targeting sequence is complementary to the RBS ofthe bacterial target gene transcript.
 3. The RNA composition of claim 1,wherein the bacterial target gene transcript is part of a polycistron.